CN106501225B - The sugar chain of agglutinin group identification is distinguishing the application in pancreatic mucinous cystic tumors and pancreas serosity cystoma - Google Patents
The sugar chain of agglutinin group identification is distinguishing the application in pancreatic mucinous cystic tumors and pancreas serosity cystoma Download PDFInfo
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses the sugar chains of agglutinin group identification to distinguish the application in pancreatic mucinous cystic tumors and pancreas serosity cystoma.The sugar chain of agglutinin group identification disclosed by the invention is by WGA, BPL, STL, DBA, sugar chain in the glycosylation albumen of the agglutinin group identification of PTL-I and MAL-I composition, these sugar chains are different in the pancreas cyst fluid of mucinous cystic tumors and serosity cystoma patient, STL, WGA, content of the sugar chain of BPL and DBA identification in MCN patient's pancreas cyst fluid is significantly higher than SCN patient, and content of the sugar chain of PTL-I and MAL-I identification in MCN patient's pancreas cyst fluid is substantially less than SCN patient, it is 0.714 combining sensitivity when distinguishing SCN patient and MCN patient with BPL using WGA, specificity is 1.Show that agglutinin group of the invention can be used for distinguishing SCN patient and MCN patient.
Description
Technical field
The present invention relates in field of biotechnology, the sugar chain of agglutinin group identification distinguish pancreatic mucinous cystic tumors and
Application in pancreas serosity cystoma.
Background technique
With the progress and extensive use of Imaging Technology, cystic pancreatic disease (pancreatic cyst lesion,
PCL) recall rate improves year by year.The nearest Research statistics PCL total prevalence rate in the U.S. is 2.5%, and cystic pancreatic disease is by one group
The heterogenous lesion for possessing the lesion composition of the different pathological types of common symptoms, mainly includes the simple false capsule of pancreas
Swollen (PPs) and cystic Tumor of Pancreas (pancreatic cystic neoplasms, PCNs).PCNs belongs to invisible morbidity early stage
Without obvious sign, tumour is formed as main feature, in all primary pancreases using glandular tube or acinar epithelia hyperplasia, ischesis
Account for about 1%~5% in adenoncus tumor.Cystic Tumor of Pancreas is subdivided into mucinous neoplasms by 2010 editions WHO stagings: mucus capsule
Property tumour (Mucinous cystic neoplasm, MCN), intraductal papilloma (Intraductal papillary
Mucinous neoplasm, IPMN) and non-mucinous neoplasms: serosity cystoma (Serous cystic neoplasm,
SCN), solid-pseudopapillary tumors (Solid pseudopaillary neoplasm, SPN.Wherein, malignant pancreatic cystic lesion packet
Include mucinous cystic tumors (MCN) and intraductal papillary-mucinous tumor (IPMN) and mucus cystadenocarcinoma (Mucinous
Cystic adenocarcinoma, MCA)；And serosity cystoma (SCN) is generally benign lesion.Different pathological types its
Treatment method is different, but single is difficult to distinguish that its is good pernicious, and cystic lesion is usually sent out by CT from clinical symptoms and Radiologic imaging
It is existing, but CT is lower than 70% for the sensibility of diagnosing malignant tumor, and specificity is then between 87%-98%, the T2WI of MRI
It is mutually capable of providing better soft tissue contrast, thus is more often available to the good pernicious antidiastole of tumour, but its accuracy is still
It stays in the lower section of 20%-80%.Single accuracy for identifying the good evil of cystic lesion from iconography is still lower, commonly uses simultaneously
Serology tumor markers: CEA；CA19-9；CA72-4 and CA153 etc., specificity, sensitivity in terms of identifying precancerous lesion
Property is lower.Histopathologic examination is still the goldstandard of current clinical diagnosis, but in view of the intrinsic defect of histological examination as damaged
The inspection of wound property is unable to dynamic detection etc., and the method for early diagnosis for seeking minimal invasive is the key that prevention and control disease canceration endoscopic ultrasonography
Lower fine needle aspiration biopsy (endoscopic ultrasonography-guided fine needle aspiration, EUS-
FNA) the intuitive firsthand information can be provided for the diagnosis of cystic Tumor of Pancreas.
Protein glycosylation is modified after a kind of most common protein translation, is to turn carbohydrate under glycosyl transferase effect
Move to the process that amino acid residue special on protein and protein forms glycosidic bond.Studies have shown that 70% human protein packet
Containing one or more sugar chains, 1% human genome takes part in the synthesis and modification of sugar chain.The glycosyl of protein in mammal
Three kinds: N- glycosylation, O- glycosylation and GPI glycosyl-phosphatidyl inositol anchor can be divided by changing type.Most sugars protein contains only
A kind of type of glycosylation, but some polypeptides are connected with N- sugar chain, O- sugar chain or ammonia polyose of candy simultaneously.
Agglutinin can specific recognition difference sugar chain structure and can be with multivalent forms in conjunction with sugar chain high-affinity due to it
Characteristic be widely used in the research that sugar group is learned, lectin chip is sugared by being fixed in the agglutinin probe on chip and sample
The specific binding of albumen sugar chain, can detect the variation of glycoprotein candy chain structure and connection type in sample with high throughput, be
Study one of most effective analysis tool of glycoprotein candy chain structure change.
Summary of the invention
The technical problem to be solved by the present invention is to how distinguish pancreatic mucinous cystic tumors and pancreas serosity capsule
Tumour.
In order to solve the above technical problems, present invention firstly provides the substances of the sugar chain of detection agglutinin group identification following
A1 the application in) or A2):
A1) preparation differentiation or supplementary globe pancreatic mucinous cystic tumors (Mucinous cystic neoplasm, MCN)
Patient and pancreas serosity cystoma (Serous cystic neoplasm, SCN) patient product；
A2) differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma；
The agglutinin group includes b1) or b2):
B1) WGA and/or BPL；
B2) by b1) with STL, DBA, PTL-I and MAL-I in the composition completely or partially formed.
In above-mentioned application, the agglutinin group may also include a kind of at least one of 30 agglutinins；Described 31
Kind of agglutinin is Jacalin, ECA, HHL, WFA, GSL- II, MAL- II, PHA-E, SJA, PNA, EEL, AAL, LTL, MPL,
LEL、GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、ConA、PTL-Ⅱ、DSA、SBA、VVA、NPL、PSA、ACA、UEA-Ⅰ、PWM、GNA、
PHA-E+L and SNA.
Wherein, the sugar chain that each agglutinin identifies in the agglutinin group is as shown in table 2 in embodiment or/and table 4.
In above-mentioned application, the agglutinin group can be only b1) or b2), can also be for by b1) or it is b2) a kind of with described 30
The composition of at least one of agglutinin composition.
In above-mentioned application, the sugar chain content of the agglutinin group identification can be the sugar chain of agglutinin group identification in pancreas
Content in cyst fluid.
In above-mentioned application, the substance of the sugar chain content of the detection agglutinin group identification may include that also can be only detection
Reagent and/or instrument needed for the sugar chain content of the agglutinin group identification.The sugar chain of the detection agglutinin group identification
The substance of content specifically may include or can be the agglutinin group or the chip containing the agglutinin group.
The reagent of the sugar chain content of detection agglutinin group identification may include the agglutinin group, fluorescent dye,
Block buffer, incubation buffer, PBST and/or PBS.The reagent of the sugar chain content of the detection agglutinin group identification
It can be only by the agglutinin group, the fluorescent dye, the Block buffer, the incubation buffer, PBST and/or PBS group
At.The fluorescent material can be Cy3 (Amerhsma company, the U.S.).The Block buffer can be for BSA, sweet be added into PBS
The solution that propylhomoserin and Tween-20 are obtained, the concentration of BSA, glycine and Tween-20 are respectively 2% in the Block buffer
(mass percent concentration), 500mmol/L and 0.1% (mass percent concentration).The incubation buffer can be to add into PBS
Enter the solution that BSA, glycine and Tween-20 are obtained, the concentration of BSA, glycine and Tween-20 point in the incubation buffer
It Wei not 2% (mass percent concentration), 500mmol/L and 0.1% (mass percent concentration).PBST is to be added to PBS
The solution that the Tween-20 mass percent concentration that Tween-20 is obtained is 0.1%.Glycine can be U.S. Sigma-Aldrich
Products.Tween-20 can be Sigma-Aldrich's product.The pH of PBS above can be 7.4.
The instrument of the sugar chain content of detection agglutinin group identification may include the chip and/or
GenPix4000B chip scanner.The sugar chain content of detection agglutinin group identification also can be only the chip and/
Or GenPix 4000B chip scanner.GenPix 4000B chip scanner is U.S. Axon Products.
The substance of the sugar chain content of the detection agglutinin group identification can also be by detecting the agglutinin group identification
Reagent needed for sugar chain content and/or instrument and data processing equipment form, and the data processing equipment is to be measured for that will come from
The content of the sugar chain of the agglutinin group identification of object is converted to the diagnostic value of the object to be measured, according to the object to be measured
Diagnostic value determine the object to be measured be MCN patient or SCN patient.The data processing equipment can be software and/or module.
The software can be 3.0 software of GenePix.
In one embodiment of the invention, elaborate in the agglutinin group WGA and BPL distinguish MCN patient and
The method of SCN patient, which comprises by the fluorescence of the content of the sugar chain of reaction WGA and BPL identification from object to be measured
Normalization numerical value substitutes into formula 1 and obtains the diagnostic value of the object to be measured；
In formula 1, WGA indicates fluorescence signal normalization numerical value when detecting the sample to be tested using WGA；BPL is indicated
The fluorescence signal normalization numerical value when sample to be tested is detected using BPL, Y indicates the diagnostic value of the sample to be tested.Formula 1
In WGA be specially fluorescence signal when WGA being utilized to detect the sample to be tested and using WGA, BPL, STL, DBA, PTL-I,
MAL-I、Jacalin、ECA、HHL、WFA、GSL-Ⅱ、MAL-Ⅱ、PHA-E、SJA、PNA、EEL、AAL、LTL、MPL、LEL、
GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、ConA、PTL-Ⅱ、DSA、SBA、VVA、NPL、PSA、ACA、UEA-Ⅰ、PWM、GNA、PHA-E
The ratio of the sum of fluorescence signal when+L and SNA detects the sample to be tested, BPL are specially to utilize BPL detection described to test sample
The fluorescence signal and utilization WGA, BPL, STL, DBA, PTL-I, MAL-I, Jacalin, ECA, HHL, WFA, GSL- II of this when,
MAL-Ⅱ、PHA-E、SJA、PNA、EEL、AAL、LTL、MPL、LEL、GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、ConA、PTL-Ⅱ、
DSA, SBA, VVA, NPL, PSA, ACA, UEA- I, PWM, GNA, PHA-E+L and SNA detect the fluorescence letter when sample to be tested
Number the sum of ratio.
Using formula 1 with the cystic Tumor of Pancreas patient of unknown MCN patient and SCN patient be object to be measured to MCN patient
When distinguishing with SCN patient, if Y is greater than 0.70, the object to be measured is or candidate is MCN patient, as Y is less than or equal to
0.70, the object to be measured is or candidate is SCN patient.
In practical applications, when distinguishing MCN patient and SCN patient using the content of the sugar chain of agglutinin group identification,
Suitable agglutinin and corresponding model can be selected to distinguish MCN patient and SCN trouble from the agglutinin group according to the actual situation
Person, as long as the content for being available with the sugar chain of agglutinin group identification of the invention distinguishes MCN patient and SCN patient,
It all belongs to the scope of protection of the present invention.
The lectin chip is that each agglutinin in the agglutinin group is fixed on the chip obtained on support.It is described
Support can be epoxide chip base.The epoxide chip base is the support prepared using epoxide.It is described
Lectin chip can contain negative Quality Control point and/or position mark.The negative Quality Control point of the lectin chip can be BSA.Institute
The position mark for stating agglutinin core can be the BSA of the fluorochrome label.Agglutinin is fixed on support using rich
Crystalline substance core SmartArrayer48 point sample instrument (Beijing Capitalbio Corporation Co., Ltd.) difficult to understand carries out.
In order to solve the above technical problems, the present invention also provides the sugar chain contents of agglutinin group identification in following A 1)
Or A2) in application:
A1) preparation differentiation or supplementary globe pancreatic mucinous cystic tumors (Mucinous cystic neoplasm, MCN)
Patient and pancreas serosity cystoma (Serous cystic neoplasm, SCN) patient product；
A2) differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma.
It is sticked in order to solve the above technical problems, the present invention also provides the sugar chains of agglutinin group identification as pancreas is distinguished
Fluidity cystoma and pancreas serosity cystoma marker are in differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas
Application in gland serosity cystoma.
In order to solve the above technical problems, the present invention also provides using the sugar chain that the agglutinin group identifies as differentiation pancreas
The differentiation pancreatic mucinous cystic tumors and pancreas serosity of mucinous cystic tumors and pancreas serosity cystoma marker
The substance of cystoma is in following A 1) or A2) in application；
A1) preparation differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma product；
A2) differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma.
In order to solve the above technical problems, the present invention also provides differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas
Gland serosity cystoma product, for the substance for detecting the sugar chain content that the agglutinin group identifies.
In the said goods, the product may include the agglutinin group or the chip containing the agglutinin group.
Chip described in the said goods can be chip described in above-mentioned application.
In the said goods, each agglutinin in the agglutinin group can be marked by fluorescent material.The fluorescent material can
For Cy3.
The substance of the sugar chain content of the detection agglutinin group identification can be the detection agglutinin group described above
The substance of the sugar chain content of identification.
In order to solve the above technical problems, the present invention also provides the products in following A 1) or A2) in application；
A1) preparation differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma product；
A2) differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma.
In order to solve the above technical problems, the present invention also provides buildings to distinguish pancreatic mucinous cystic tumors and pancreas slurries
The method of property cystoma.
The method that pancreatic mucinous cystic tumors and pancreas serosity cystoma are distinguished in building provided by the present invention, packet
It includes: the sugar chain in pancreatic mucinous cystic tumors group and pancreas serosity cystoma group pancreas cyst fluid quantitatively being divided respectively
Analysis obtains cystic Tumor of Pancreas sugar chain characteristic spectrum based on the analysis results；The cystic Tumor of Pancreas sugar chain characteristic spectrum is by described solidifying
The sugar chain composition of collection element group identification.
In the above method, the pancreatic mucinous cystic tumors group may include that at least one pancreatic mucinous cystic tumors is suffered from
Person.The pancreas serosity cystoma group may include at least one pancreas serosity cystoma patient.
In the above method, quantitative analysis is carried out to the sugar chain in pancreas cyst fluid, is carried out using the agglutinin group.It is described
Each agglutinin in agglutinin group can be marked by fluorescent material.The fluorescent material can be Cy3.
In the above method, carrying out quantitative analysis to the sugar chain in pancreas cyst fluid may include utilizing to contain the agglutinin group
Chip respectively quantifies the sugar chain in pancreatic mucinous cystic tumors group and pancreas serosity cystoma group pancreas cyst fluid
Analysis.Specifically can include: be incubated for after marking the protein in sample to be tested with the fluorescent material with the chip, determine institute
State the content of the sugar chain of the identification of agglutinin group described in sample to be tested.
The cystic Tumor of Pancreas sugar chain characteristic spectrum can be formula 1.
Pancreatic mucinous cystic tumors and pancreas serosity cystoma can be distinguished using cystic Tumor of Pancreas sugar chain characteristic spectrum.
In another embodiment of the present invention, quantitative analysis is carried out including the use of described solidifying to the sugar chain in pancreas cyst fluid
Collection element group respectively determines the sugar chain in pancreatic mucinous cystic tumors group and pancreas serosity cystoma group pancreas cyst fluid
Amount analysis.
In the present invention, the sugar chain is sugar chain in glycosylation albumen.
The difference for the glycoprotein candy chain content that the present invention is identified by comparing SCN patient's agglutinin different from MCN patient's,
And by correlation analysis, the sugar chain that six kinds of agglutinins for distinguishing SCN patient and MCN patient identify is found, this six kinds solidifying
Collection element is respectively STL, WGA, BPL, DBA, PTL-I and MAL-I, STL, WGA, BPL and DBA every kind of agglutinin of these four agglutinins
Identify that content of the sugar chain in MCN patient's pancreas cyst fluid is significantly higher than SCN patient, and both agglutinins of PTL-I and MAL-I are every
Content of the kind agglutinin identification sugar chain in MCN patient's pancreas cyst fluid is substantially less than SCN patient, is utilizing WGA and BPL association area
Dividing sensitivity when SCN patient and MCN patient is 0.714, specificity 1.Show the sugar chain of agglutinin group identification of the invention
It can be used as biomolecule mark, for distinguishing SCN patient and MCN patient, can use agglutinin group and its knowledge of the invention
Other sugar chain distinguishes SCN patient and MCN patient.
Detailed description of the invention
Fig. 1 is lectin chip sample application array figure and the fluorescence detection figure applied to glycoprotein candy chain, and A is agglutinin in figure
Chip sample application array figure, B are pancreas Mucinous cystoadenoma (MCN), pancreatic serous cystadenoma (SCN) patient's cystic Tumor of Pancreas
Cyst fluid glycoprotein candy chain fluoroscopic examination result figure；
Fig. 2 is GenePix3.0 software analysis chip scanning figure, and wherein abscissa indicates fluorescence intensity.
Fig. 3 is that ROC curve analyzes result.
Fig. 4 is the proof diagram by taking STL and BPL as an example.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Block buffer in following embodiments is the solution that addition BSA, glycine and Tween-20 are obtained into PBS,
In Block buffer the concentration of BSA, glycine and Tween-20 be respectively 2% (mass percent concentration), 500mmol/L and
0.1% (mass percent concentration).Incubation buffer is the solution that addition BSA, glycine and Tween-20 are obtained into PBS,
In incubation buffer the concentration of BSA, glycine and Tween-20 be respectively 2% (mass percent concentration), 500mmol/L and
0.1% (mass percent concentration).PBST is that the Tween-20 mass percent concentration that Tween-20 is obtained is added to PBS to be
0.1% solution.Glycine can be Sigma-Aldrich's product.Tween-20 can be U.S. Sigma-Aldrich
Products.The pH of PBS above is 7.4.
Embodiment 1, the glycosylation albumen sugar chain for distinguishing MCN patient Yu SCN patient
(1) research object and sample collection
Research object: through Hospital Ethical Committee by (ethics number: S2014-108-01), malignant tumour medical history is excluded；
Serious cardiopulmonary insufficiency；Bleeding and clotting obstacle；Early period is clear through imaging diagnosis, Chinese People's Liberation Army General Hospital's digestion
Internal medicine combination tumor surgery lesion accepts PCNs patient for medical treatment, wherein the MCN patient made a definite diagnosis 17 (entering a group MCN group), SCN patient 18
(entering a group SCN group).
Cyst fluid acquisition at least 0.2ml is simultaneously immediately placed on ice, and protease inhibitors is added, and (1 μ is added in every 0.1 milliliter of cyst fluid
L protein degradation) is prevented.
(2) processing of cystic Tumor of Pancreas cyst fluid albumen and fluorescent marker
35 cyst fluid samples respectively take respective volume (including 100 μ g total proteins) and 100 μ L 0.1M Na2CO3(pH 9.3) is slow
Fliud flushing mixes, and mixed liquor and Cy3 fluorescent dye are incubated at room temperature 3h, the albumen marked with Sephadex G-25 column separating purification Cy3
Matter, and in spectrophotometric determination collection liquid be labeled protein concentration.
(3) lectin chip and data analysis
The agglutinin group of 37 kinds of agglutinins composition, the sample application array of agglutinin A institute as shown in figure 1 are fixed on lectin chip
Show, the details of each agglutinin are as shown in table 1.
37 kinds of agglutinins in table 1 are made into according to the operation instruction that each agglutinin producer provides to the point sample of 1mg/mL respectively
Liquid.Wherein Cy3 label and the BSA of non-marked be respectively position mark and negative Quality Control point.By rich brilliant core difficult to understand
Every kind of agglutinin of parameter is arranged in SmartArrayer48 point sample instrument (Beijing Capitalbio Corporation Co., Ltd.) 48 spotting systems
Repeat point sample three times, the array that point sample matrix is 4 × 10 repeats 4 areas of point system, and point is formed in epoxide chip base.Point sample
It finishes for chip to be placed in the environment that humidity is 50% and be incubated overnight, then the dry 3h in 37 DEG C of vacuum ovens, convenient for solidifying
Collection element is effectively combined with chip base, spare.Before closing, puts the chip made and respectively clean core with 1 × PBST of pH7.4 and 1 × PBS
Piece 2 times, each 5min, drying obtains lectin chip.
Table 1, lectin chip are aggregated prime information
In chip hybridization box, 4rpm at the uniform velocity rotates 25 DEG C of closing 1h in Block buffer, with PBST, the PBS of pH7.4
It respectively washes 2 times, each 5min, dries；Protein after MCN group and SCN group example sample respectively take 5 μ g to mark is buffered with incubation respectively
After liquid mixing, it is incubated at room temperature 3h with chip, is respectively washed 2 times, each 5min with 10mmol/L PBST and 10mmol/L PBS, is dried；
GenPix 4000B chip scanner (Axon company, the U.S.) scans chip.Then use 3.0 software of GenePix from scanning result
The information such as fluorescence signal intensity value and background value are obtained in figure to be analyzed, and can get every on MCN group and SCN group agglutinin chip
The fluorescence signal value of a point, the corresponding 3 repetition points of every kind of agglutinin in this chip, i.e., practical every kind of agglutinin repeat detection 3
It is secondary, it obtains 3 signal datas, extracts intermediate value, i.e., each corresponding intermediate value of agglutinin.Calculate the intermediate value institute of each agglutinin
The ratio for accounting for the sum of 37 kinds of agglutinin intermediate values completes data normalization.Each sample is done to be repeated three times.Agglutinin normalizes fluorescence
Intensity is expressed as duplicate mean value ± standard deviation value three times.It calculates each agglutinin fluorescence intensity and normalizes numerical value in MCN group
The opposite variation of glycosylated protein compared with coming with the ratio (i.e. ratio value) of SCN group normalization numerical value.Data utilize simultaneously
SPSS19 carries out t check analysis.
The result of the GenPix 4000B chip scanner of one MCN group sample and SCN group sample scanning chip is such as
In Fig. 1 shown in B.GenePix3.0 software analysis chip scanning figure is as shown in Figure 2.The result shows that in (1) MCN group and SCN group,
It glycosylates in albumen, Jacalin, LEL, the signal strength mean value of LCA, ConA and ACA are all larger than 0.05, show its specificity
For the sugar chain of the glycosylation albumen of identification based on high mannose and core fucose sugar chain structure, the height of LEL and ConA identification is sweet
Reveal sugar-type N- sugar chain, the Gal β 1-3GalNAc α-Ser/Thr (T) and GalNAc α-Ser/Thr (Tn), ACA of Jacalin identification
The Gal β 1-3GalNAc α-Ser/Thr (T antigen) of identification, content is higher in two class cystadenoma cyst fluids；(2) it is aggregated
The terminating in GalNAc α/β 1-3/6Gal structure of plain WFA identification, the trimers and of STL identification
The N- sugar chain structure of tetramers of GlcNAc and core (GlcNAc) only have high level (3) in MCN group sample
And the GalNAc, GalNAc α -1,3Gal, GalNAc α -1,3Gal β -1,3/4Glc of agglutinin PTL-I identification, agglutinin MAL-I
Only content increases the Gal β -1,4GlcNAc structure of identification in SCN group sample.
The variation of MCN, SCN cyst fluid glycoprotein candy chain spectrum:
MCN group and SCN group cyst fluid sample are detected respectively using lectin chip, chip data is obtained by software
And after normalized, ratio value (table 2, table 3 and table 4) is calculated, normalization data is as shown in Figure 2.
The agglutination of the sugar chain structure of the glycoprotein candy chain structure and specific recognition of differential expression in table 2, MCN and SCN cyst fluid
Element
Table 3, each sample fluorescence signal normalize numerical value
The Ratio value of table 4, remaining agglutinin
According to statistical theory, Ratio value between 0.5 and 2, then in sample identical glycoprotein candy chain content in MCN
Indifference between two groups of samples of group and SCN group；Value >=2 Ratio, then the glycoprotein candy chain content in MCN group sample is significantly higher than SCN
Identical glycoprotein candy chain content in group；Value≤0.5 Ratio, then the glycoprotein candy chain content in MCN group sample is substantially less than SCN
The content of identical glycoprotein candy chain in group.
As a result, it has been found that the glycosylation albumen sugar chain content of 6 kinds of agglutinins identification has significantly in MCN group and SCN group cyst fluid
Difference.In glycoprotein candy chain, the N- of trimers and tetramers of GlcNAc and core (GlcNAc) of STL identification
Gal β 1-3GalNAc and Terminal GalNAc, the DBA that sugar chain structure, the multivalence sialic acid structure of WGA identification, BPL are identified
The α GalNAc of identification, Tn antigen, GalNAc α 1-3 ((Fuc α 1-2)) Gal (blood group A antigen) are in MCN
Content obviously increases in group.The sugar chain structure of both agglutinins of PTL-I and MAL-I identification content in SCN group dramatically increases
(R < 0.5, p < 0.05).Show to can use this 6 kinds of agglutinins and glycoprotein candy chain containing its identification distinguish MCN patient with
SCN patient.
To distinguish MCN patient and SCN patient, constructs by taking WGA and BPL as an example and constructed using Influencing factors
The mathematical model for distinguishing MCN patient and SCN patient, is shown in formula 1.
In formula 1, sample fluorescence signal normalization numerical value when WGA expression is detected using WGA, BPL indicates to utilize BPL
Sample fluorescence signal normalization numerical value when being detected, Y indicate the diagnostic value of sample.
Using formula 1 with the cystic Tumor of Pancreas patient of unknown MCN patient and SCN patient be object to be measured when carry out ROC
Tracing analysis (Fig. 3), area under the curve AUC=0.853, sensitivity 0.714, specificity 1, this method is determined as cancer of pancreas
The threshold value of patient is 0.70, i.e., when Y is greater than 0.70, doubtful object to be measured is MCN patient, when Y is less than or equal to 0.70, to
Survey object is SCN patient.
(4) agglutinin trace
For further verify lectin chip as a result, by taking agglutinin STL and BPL as an example, by the agglutination of Cy3 fluorescent marker
Element is incubated for the cystic Tumor of Pancreas cyst fluid albumen after transferring film, is analyzed through scanner scanning and 3.0 software of Genepix, and determination contains
There is content situation of the glycosylation albumen of the sugar chain of STL and BPL identification in MCN group and SCN group cyst fluid.Concrete operation step is such as
Under:
By 30 μ g albumen samples first through PAGE gel electrophoresis, after electrophoresis, gel is taken out, in transferring film buffer
Rinse the several seconds.Pvdf membrane using it is preceding first pre-process 5min with anhydrous methanol after go in transferring film buffer balance it is good after use.It takes
Gel is using the half-dried transferring film instrument of TE 70PWR according to pvdf membrane electric current 0.8mA every square centimeter, constant current transferring film 1.5h out.Transferring film is complete
Bi Hou is placed into pvdf membrane in preprepared TBST cleans twice immediately, and each 10min washes away remaining transferring film on film
Then buffer is transferred in Carbo-free confining liquid, jog 1h is closed on shaking table.After closing, directly by Cy3 fluorescence
The agglutinin of label is added in Carbo-free confining liquid by 2 μ g/mL of final concentration, is protected from light overnight for 4 DEG C of jog on shaking table.TBST
Cleaning film 3 times, each 10min, it is 800 that PMT is then arranged in Storm840 gel imaging system, red fluorescence channel
Scan image under (635nm excitation wavelength/650LP launch wavelength) reads protein band gray value with Image J software.
As a result (Fig. 4) is shown, the fluorescence intensity of STL and BPL in the cyst fluid sample of 18 MCN patients is apparently higher than 17
Fluorescence intensity in example SCN patient's cyst fluid sample shows to glycosylate in albumen, and the sugar chain that STL and BPL are combined is in MCN patient's capsule
Content in liquid is higher than the content in SCN patient's cyst fluid.At two protein bands (white edge sketches the contours) of 60kDa and 50kDa,
STL is significantly higher than the fluorescence intensity (P < 0.05) in SCN patient as fluorescence intensity of the probe in MCN patient；?
At the protein band (white edge sketches the contours) of 120kDa, BPL is significantly higher than as fluorescence intensity of the probe in MCN patient and suffers from SCN
Fluorescence intensity (P < 0.05) in person.It is consistent with lectin chip result as the result is shown.Prove that these agglutinins can be used as spy
Needle distinguishes SCN patient and MCN patient by the detection to the glycoprotein candy chain in cystic Tumor of Pancreas cyst fluid.
Claims (7)
1. detecting the substance of the sugar chain content of agglutinin group identification in preparation differentiation or supplementary globe pancreatic mucinous cystic tumors
Application in patient and pancreas serosity cystoma patient product:
The agglutinin group includes WGA, BPL, STL, DBA, PTL-I and MAL-I.
2. application according to claim 1, it is characterised in that: the agglutinin group further include Jacalin, ECA, HHL,
WFA、GSL-Ⅱ、MAL-Ⅱ、PHA-E、SJA、PNA、EEL、AAL、LTL、MPL、LEL、GSL-Ⅰ、LCA、RCA120、BS-Ⅰ、
ConA, PTL- II, DSA, SBA, VVA, NPL, PSA, ACA, UEA- I, all or part in PWM, GNA, PHA-E+L and SNA.
3. application according to claim 1 or 2, it is characterised in that: the sugar chain content of the agglutinin group identification is described
Content of the sugar chain of agglutinin group identification in pancreas cyst fluid.
4. using the sugar chain that agglutinin group described in claims 1 or 2 identifies as differentiation pancreatic mucinous cystic tumors and pancreas
It is prepared by the differentiation pancreatic mucinous cystic tumors of serosity cystoma marker and the substance of pancreas serosity cystoma
Application in differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma product.
5. differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas serosity cystoma product, to detect claim
The substance of the sugar chain content of the identification of agglutinin group described in 1 or 2.
6. product according to claim 5, it is characterised in that: the product includes agglutinin described in claims 1 or 2
Group or chip containing agglutinin group described in claims 1 or 2.
7. product described in claim 5 or 6 is in preparation differentiation or supplementary globe pancreatic mucinous cystic tumors and pancreas slurries
Application in property cystoma product.
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| CN110045126B (en) * | 2019-04-03 | 2022-08-09 | 中国医学科学院北京协和医院 | Biomarker for diagnosing autoimmune pancreatitis and application thereof |
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| CN111220804B (en) * | 2020-01-19 | 2023-04-28 | 深圳格道糖生物技术有限公司 | Marker for evaluating breast cancer chemotherapy effect based on serum glycoprotein |
| CN111551723A (en) * | 2020-06-01 | 2020-08-18 | 深圳格道糖生物技术有限公司 | Application of specific lectin in identification of pancreatic cancer based on salivary glycoprotein carbohydrate chain structure and related products |
| CN112067822B (en) * | 2020-08-31 | 2023-04-18 | 西北大学 | Application of lectin probe combination in aspect of identifying sex of black-leaf monkeys based on urine protein glycoform |
| CN113702637B (en) * | 2021-08-09 | 2023-04-07 | 西北大学 | Lectin test carrier, kit and prediction model for predicting neoadjuvant chemotherapy curative effect of breast cancer |
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